The synthesized CEAapt-TDN-MOF is a very stable detection system due to the vertex phosphorylated TDN structure at the interface, possessing a one-year shelf-life. Moreover, it exhibits a significant horseradish peroxidase mimicking activity due to the iron porphyrin ring, which leads to a colorimetric reaction upon binding toward antibody-captured CEA. Using this method, we successfully achieved the highly specific and ultra-sensitive detection of CEA with a limit of detection as low as 3.3 pg/mL. In addition, this method can detect and analyze the target proteins in clinical serum samples, effectively identify the difference between normal individuals and patients with colon cancer, and provide a new method for the clinical diagnosis of tumors, demonstrating a great application potential.Carcinoembryonic antigen (CEA) is an important broad-spectrum tumor marker. For CEA detection, a novel type of metal–organic framework (MOF) was prepared by grafting CEA aptamer-incorporated DNA tetrahedral (TDN) nanostructures into PCN-222 (Fe)-based MOF (referred as CEA The metal–organic framework (MOF) has been widely used in the fields of catalysis, separation, and sensing due to its unique crystalline porosity, flexible tailorability, and large specific surface area. Post-synthetic modification of MOF is mainly used to adjust the physical and chemical properties of the porous framework structure, so as to endow the MOF with specific functions for a variety of application fields ,2. EspecTo overcome the aforementioned limitations, Wang et al. recently reported a facile and efficient post-modification strategy by using the strong interaction between the phosphate group of dihydroxyphenylalanine (DOPA) and the metal site of zirconium MOF. The nano-MOFs crystals can be dispersed in various chemical solvents in the form of colloids . Based oapt-TDN-MOF). Specifically, in order to achieve a good molecular recognition and interface assembly process, zirconium metal ions were used as ligands so as to realize high-efficiency coordination assembly with vertex phosphorylation-modified DNA tetrahedron, as well as the successful integration of a CEA aptamer sequence in a TDN structure for capturing targets. In addition, to achieve signal amplification, MOFs were endowed with very strong horseradish peroxidase mimicking activity by using iron tetrabenzoate porphyria. Meanwhile, a proteoglycan protein CEA, one of the most frequently detected tumor markers for early diagnosis of colon cancer, was chosen as our target [apt-TDN-MOF could efficiently detect it by exhibiting a significant colorimetic signal. In this work, we designed and prepared DNA tetrahedron nanostructure (TDN)-functionalized MOF colloid nanorods as a colorimetric biosensor for the sensitive detection of the tumor marker carcinoembryonic antigen (CEA)  was used to observe the structure and morphology of the MOF material PCN-222 (Fe), as shown in apt-TDN-MOF was successfully prepared. Moreover, the prepared CEAapt-TDN-MOF might efficiently detect its target molecule.In order to further explore the successful modification of TDN on the MOF nanorods, the D chain was replaced by an E chain with a large number of T base sequences at the end, and we assembled a new tetrahedral structure of TDN’ with four single strands of A, B, C, and E DNA. At the same time, the I chain with a multi-A base sequence was grafted on gold nanoparticles with a size of about 13 nm to prepare DNA-AuNPs. Using the complementary pairing of the sequence of A-T bases, a strong molecular recognition event could occur between DNA-AuNPs and the newly prepared TDN’-MOFs. As shown in apt-TDN-MOF remained 27% of the maximum activity , and TMB/H2O2/CEAapt-TDN-MOF solutions and the corresponding color response results. There was almost no color change in the solutions of TMB and TMB/H2O2 (a and b), but there was an obvious blue change in the solution after the addition of PCN-222 (Fe) (c), indicating that the PCN-222 (Fe) had a very good catalytic oxidation activity. Under the same reaction conditions, the reaction solution of CEAapt-TDN-MOF (d) also changed obviously blue. To intuitively compare the differences between each group, the UV absorption spectra of TMB in each group’s solution was recorded at a wavelength range of 550–700 nm (with a peak at 652 nm), and the results remained in accordance with the color reaction changes. The group a and group b spectra were similar, showing no UV absorption peak at this wavelength range. Meanwhile, both group c and group d had very high peaks, indicating the high completion of catalytic reaction. As illustrated in apt-TDN-MOF via Fenton reaction: Fe2+ + H2O2 3+ + HO·+ OH−. Moreover, group d had a slightly lower peak than group c but almost negligible, indicating that TDN functionalization had very little effect on the catalytic activity of PCN-222 (Fe), which could effectively guarantee the feasibility of the experiment. In addition, the catalytic ability of CEAapt-TDN-MOF was studied by recording the time-varying absorbance curve of TMB at 652 nm. As shown in apt-TDN-MOF increased with the increase of concentration, and the corresponding concentrations of each curve were 0, 6.25, 12.5, 25, 35, and 50 μg/mL (a to f), respectively. Therefore, the catalytic performance of the system could be achieved by adjusting the concentration of CEAapt-TDN-MOF in practical applications. Moreover, it was found that this CEAapt-TDN-MOF exhibited superior catalytic effect compared with nature horseradish peroxidase , where the CEAactivity . To inveroxidase , indicatapt-MOF for the colorimetric sensing of CEA. The results show that the signal response value of CEAapt-TDN-MOF was higher than that of CEAapt-MOF under the same conditions (CEA concentration is 20 ng/mL), which was probably because the rigid structure of TDN could effectively promote the molecular recognition between the aptamer sequence and target protein, while the flexible single-strand structure might adhere to the surface of the MOF material and reduce the binding efficiency by 35% [apt-TDN-MOF with respect to CEAapt-MOF (showing an obvious downward trend in detection ability from the sixth month)  containing a CEA aptamer was grafted on the surface of PCN-222 (Fe) to prepare CEAy by 35% . Moreoveh month) . Meanwhiapt-TDN-MOF exhibited a good linear detection range of 0.01–25 ng/mL, and the detection limit was 3.3 pg/mL  even at the concentration of 100 ng/mL. These results demonstrate that our colorimetric sensing system has very high sensitivity and specificity  materials with simulated horseradish peroxidase activity and constructed a colorimetric immune biosensor for the ultra-sensitive detection of CEA. The phosphorylated DNA tetrahedral structure could be effectively fixed on the PCN-222 (Fe) surface through strong coordination with zirconium ions, thus bringing very stable performance. Moreover, the rigid structure of TDN greatly promoted the binding of the aptamer sequence at the end of the tetrahedron to the target. As a consequence, the strong molecular recognition ability and high enzyme catalytic activity ensure that the colorimetric sensing method has very high specificity and sensitivity, and the detection limit is as low as 3.3 pg/mL , Transmission Electron Microscopy , X-ray Powder Diffractometer , Milli-Q ultrapure water purification system , zeta potential analyzer , UV spectrophotometer , and multifunctional microplate reader  were applied.N,N-dimethylformamide (DMF) and its chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich . 1 × PBS (containing 0.05% Tween-20) solution, blocking buffer (BSA), and universal antibody dilutions were purchased from Shanghai SANGON biotech Co., Ltd. . Human carcinoembryonic antigen (CEA) and CEA antibody were purchased from Shanghai LingChao Bio Technology Co., Ltd. . A Human CEA ELISA Kit  was purchased from Thermo Fisher . Gold nanoparticles (AuNPs) with a diameter of 13 nm were purchased from Jiangsu Shengfeng nanomaterials technology Co., Ltd. . The 96-well plates were purchased from Corning . Water used throughout the experiment was ultrapure water . The remaining chemicals were of analytical grade and used without further purification. All oligonucleotide strands were synthesized by the Shanghai bioengineering company and subjected to HPLC purification. DNA sequences are shown in Zirconia dichloride octahydrate and meso-tetra (4-carboxyphenyl) porphine ferric chloride (Fe-TCPP) were purchased from Shanghai Lingwei Reagent Co., Ltd. . First, 38 mg of zirconia dichloride octahydrate was dissolved with 6.5 mg of Fe-TCPP in 16.3 mL of DMF and sonicated for 1 min to mix well. Then, 0.25 mL of dichloroacetic acid was added into the mixed solution, transferred into a Teflon-lined hydrothermal reaction tank, and tightened to sit in a 180 °C oven for 18 h to heat. After the reaction, the as-synthesized nanocrystals were obtained by the method of centrifugation , which was immediately followed by centrifugal washing with DMF and ethanol solution to remove the precursor species that did not participate in the reaction, and repeated 3 times. Finally, the solid was dried under vacuum at 80 °C overnight for further use.2, pH 8.0) at equal ratios. Then, the solution was heated to 95 °C and re-cooled to 4 °C to complete the renaturation process. In addition, the preparation of TDN-bearing poly-T sequences  was similar to the procedure described above. Finally, the as-prepared two tetrahedron structures were stored at 4 °C for further use.The preparation process of TDN with aptamer sequences was described as following: four single-stranded DNAs  were mixed in TM buffer  concentration was 1 μM.First, 0.2 mL solution containing TDN (10 μM) was added to 2 mg (783 nmol) PCN-222 (Fe) nanorods and incubated on a shaker for 4 h. During this period, the NaCl solution was added slowly to reduce the electrostatic repulsion, and the final concentration was 0.5 M. Then, the excess nucleic acid structure was removed by centrifugal washing , and the operation was repeated three times. Finally, it was re-dispersed into TM buffer and stored at 4 °C for use.g) at 4 °C, and resuspended in 1 mL of 10 nM phosphate (PB) buffer. In addition, 100 μM of thiol-modified DNA sequence (strand J) was activated by incubation with 10 nM of TCEP solution for 1 h. Then, 35 μL of activated DNA was added to the above gold nanoparticles solution  and incubated for 16 h. Afterwards, 9 μL of NaCl solution (2 M) was gradually added to the reaction system, and the final salt concentration was 0.1 M, during which we were gently tapping the wall to avoid aggregation. The DNA-modified gold nanoparticles were centrifuged at 4 °C for 15 min  to remove the supernatant and resuscitated with 10 mM PB buffer. Then, 500 nmol PCN-222 (Fe) nanorods decorated with TDN with poly-T sequences were incubated with DNA-modified gold nanoparticles for 2 h to complete the assembly behavior, and finally, DNA-modified gold nanoparticles without the assembly behavior occurring were removed by centrifugation.First, 1 mL of 40 nm diameter gold nanoparticles was taken from the mother liquor into a low-adsorption EP tube, centrifuged for 15 min  was added with gentle shaking for 20 min, after which 50 μL of stop solution (1 M concentrated sulfuric acid) was added to stop the reaction, and the absorbance intensity was measured at 450 nm with a microplate reader. The standardized ELISA test was performed by following the instructions for the product: EHCEA (96 tests) at thermofisher.com, accessed on 18 October 2021.First, 100 μL of capture antibody anti-CEA solution (3 μg/mL) was added to each well of a 96-microwell plate and incubated at 4 °C overnight. After the antibody solution was removed, the antibody solution was washed with PBST buffer. Then, 150 μL BSA sealing solution was added to each well. After incubation at 37 °C for 1 h, it was washed 3 times with PBST buffer. Then, each well was incubated with different concentrations of the target protein (CEA) 37 °C for 1 h, the well with only PBST solution was set as the blank control group, and the washing step was repeated three more times. This was immediately followed by the addition of 100 μL of TDN-functionalized modified PCN-222 (Fe) nanorods ) to each well, and the wells were sealed with a sealing film and incubated at room temperature for 1 h. Finally, 100 μL of TMB substrate solution (2 mM TMB and 0.2 M HHuman serum samples from colon cancer patients were obtained from the Second Affiliated Hospital of Nanjing, Southeast University. Informed consent was obtained from all cases, and this study was approved by the Scientific and Ethical Committee of Nanjing University and Southeast University. The serum sample detection chamber simply needs to exchange the target solution for a 100 μL serum samples, and the other processes are similar to the above steps.


The fluorescence spectra and CD spectra showed that sterilization caused a decrease in the content of α-helix and β-sheet and a significant increase in β-turn (p < 0.05). Sterilization changed the EWP-Ca structure and decreased its stability; the calcium-binding capacity of EWP-Ca after sterilization was decreased to 41.19% (p < 0.05). Overall, these findings showed that EWP could bind with calcium, form a peptide–calcium chelate, and serve as novel carriers for calcium supplements.With the current study, we aimed to determine the characteristics and calcium absorption capacity of egg white peptide–calcium complex (EWP-Ca) and determine the effect of sterilization on EWP-Ca to study the possibility of EWP-Ca as a new potential calcium supplement. The results of SEM and EDS showed a high calcium chelating ability between EWP and calcium, and the structure of EWP-Ca was clustered spherical particles due its combination with calcium. The FTIR and Raman spectrum results showed that EWP could chelate with calcium by carboxyl, phosphate, and amino groups, and peptide bonds may also participate in peptide–calcium binding. Moreover, the calcium absorption of EWP-Ca measured by the intestinal everted sac model in rats was 32.38 ± 6.83 μg/mL, significantly higher than the sample with CaCl Eggs are rich, high-quality protein foods essential for daily living . Due to In the human body, calcium is one of the richest inorganic elements. It accounts for 1.5–2.2% of the human body weight  and is iThe purpose of this research was to prepare an EWP-calcium complex (EWP-Ca) and investigate its characterizations, ability to promote calcium absorption, and stability during the sterilization process. We anticipate that these findings will provide a theoretical basis for the potential use of EWP-Ca as a new type of calcium supplement.Egg white powders were obtained from Heilongjiang ZhongNongXingHe Biotechnology Co., Ltd. . The chemicals in our experiment were of analytical grade.w/v) and heated at 90 °C for 30 min, then hydrolyzed the solution by Alcalase (3% w/v) at 50 °C for 3 h at pH 10.66. After that, the enzyme in the solution was inactivated for 10 min through a boiling water bath, followed by centrifugation , and the supernatant was collected for freeze-drying and obtaining the EWP powders.The preparation of egg white peptide (EWP) was performed as described by Lin et al.  with som2 to the solution and made the mass ratio of EWP to calcium at 3:1 w/w. This solution was mixed by stirring at 50 °C and pH 8 for 60 min, and then ethanol (85%) was added to remove the unbound calcium. Finally, the solution was centrifuged . We collected the supernatant for freeze-drying and the obtained product was named the EWP-calcium complex (EWP-Ca).The EWP-calcium complex preparation was carried out following Sun et al. . First, Scanning electron microscope analysis was performed using the previously described method . The samFourier transform infrared (FTIR) spectra of samples were evaluated using an FTIR spectrometer  as described previously .The Raman spectra of EWP and EWP-Ca were evaluated using the Horiba LabRAM HR Evolution micro-spectrometer  as described previously .2) in the buffer at 4℃. The buffer was a mixed solution of 136 mmol/L NaCl, 8.17 mmol/L KCl, 1.0 mmol/L MgCl2, 11.1 mmol/L glucose, and 20 mmol/L HEPES. Finally, the intestine was turned over and the buffer solution was injected into the drug-free, ligation end of the small intestine.The intestinal everted sac model in rats was set up following Hou et al. . Wistar 2), casein phosphor peptide calcium (CPP–Ca), and a mixture of EWP and Ca (EWP + Ca) were used as control groups. The CaCl2, CPP-Ca, EWP + Ca, and EWP-Ca were dissolved in the buffer at 25 °C and we kept the calcium content at 2 mmol/L. We immersed the intestinal everted sac into the sample buffers and incubated them under the condition of continuous oxygen for 2 h. Then, the solution in the intestinal sac was collected, and the calcium content was evaluated by the atomic absorption method.Anhydrous calcium chloride .The composition of EWP-Ca was analyzed by Nano LC-ESI-MS/MS as described by Wang . Sample The sterilization conditions for this experiment were determined under 85 °C for 10 s, following standard market EWP production procedures. Unsterilized EWP-Ca was the control group.Fluorescence spectra were evaluated by the Hitachi F-2700 fluorescence spectrometers . We prepared the samples (0.1 mg/mL) by deionized water and measured the following parameters: excitation wavelength, 295 nm; scanning range, 295–330 nm; and slit width, 5 nm, for both excitation and emission.Circular dichroism (CD) spectra were evaluated by Jasco J-1500 spectropolarimeter  as described by Bao et al. . Samples× g, 4 °C, 5 min) and pellets were collected and dried in a vacuum freeze-dryer to determine the calcium content by atomic absorption method and calculate the calcium-binding capacity.After sterilization, absolute ethanol was added to the EWP-Ca solution (10 mL) to remove the unbound calcium, making the final concentration 85%. The solution was centrifuged , and the statistical significance was set at Microstructures of EWP and EWP-Ca are displayed in The The EDS analysis is usually used to analyze the chemical composition of samples . As show–1300 cm−1, used to evaluate the types and changes of groups, and the other one is the fingerprint region in the range of 1300–600 cm−1, used to indicate molecular characteristics and structure [–1600 cm−1 and the amide-II-vibration at 1600–1500 cm−1 were also two crucial amide vibration modes caused by the stretching and the deformation of the C-O, N-H, and C-N [The formation and composition of organic functional groups could be measured by FTIR spectroscopy, such as C-O, N-H, and O-H . It is wtructure . Further and C-N . Figure −1 was moved to 3425.78 cm−1. The change in the characteristic peak may be due to the formation of coordination bonds between nitrogen atoms and calcium ions by offering their electron pairs [−1 peak, the EWP absorption band was characterized as an amide I band caused by the C=O stretching vibration. After calcium chelation, the band corresponding to the amide I group was reduced to 1644.46 cm−1, indicating that the C=O group was involved in binding peptide and calcium. Meanwhile, the peak intensity at 1400.93 cm−1, characterized as the absorption peak of -COO-, moved to a higher peak of 1411.16 cm−1 after being chelated with calcium, indicating that COO- may be combined with Ca2+ and transformed into -COO-Ca. Furthermore, the peak at 1074.78 cm−1 of EWP may be due to the symmetrical stretching vibration of –PO3. It moved to a higher peak of 1077.83 cm−1 after being chelated with calcium, indicating that the phosphate groups may also be involved in calcium binding.Many signals could be found in the FTIR spectrum. As usual, the first part was probably caused by strong O-H and N-H stretching vibrations, where the broadband is due to hydrogen-bonded O-H groups . As showon pairs . HydrogeFTIR spectroscopy was usually used to report the chemical structures of the samples, particularly in the interaction of metal ions and organic ligand groups in peptides ,36. The −1 is mainly caused by the stretching vibration of C=O, and the peak at the position of 1330 cm−1 corresponds to the symmetrical stretching vibration of the COO- of the amino acid side chain [−1 represents α-helix, and the peak at 1670 cm−1 represents β-sheet [Information about the structure and conformation of biomolecular can be obtained through Raman spectrum analysis . As showde chain ; When th β-sheet . It can 2 in this experiment. As shown in 2 (19.93 ± 2.84 μg/mL) (p < 0.05), while higher than the EWP + Ca (24.14 ± 2.43 μg/mL) (p < 0.05), although it had no significant difference with CPP-Ca (29.56 ± 3.94 μg/mL) (p > 0.05). This revealed that the EWP-Ca and CPP-Ca had similar effects which could promote the absorption of calcium.The intestinal everted sac model in rats was used to simulate the intestinal environment in vivo and evaluate the absorption of active substances. As a popular calcium supplement product of protein peptide–calcium product on the market, CPP–Ca was used as the control group together with CaClA high-resolution mass spectrometer was used to measure EWPs which could chelate calcium. As shown in The tryptophan residue at 295 nm excitation produces fluorescence intensity at a wavelength within the range of 300–400 nm, with the potential energy of tryptophan fluorescence bands indicating a change in position of the tryptophan residue in the protein and changes in the protein structure . As show2+ and egg white peptide cause the egg white peptide to maintain a specific spatial conformation [2+ and egg white peptides. Thus, the thermal stability of egg white peptide calcium chelate was affected.CD spectroscopy is typically used to measure the secondary structures of proteins and peptides, which is an established and valuable technology ,45. FiguAs described in p < 0.05). It can be preliminarily inferred that EWP-Ca was unstable under high-temperature conditions. High temperatures destroyed the structure of the peptide, resulting in the destruction of the peptide–calcium binding bond and the release of calcium. However, high-temperature treatment did not wholly dissociate calcium ions, indicating that the bond between calcium and peptide has a certain tolerance to high temperature.After sterilization, the calcium-binding capacity of EWP-Ca significantly decreased to 41.19% (In conclusion, we found that EWP-Ca can effectively improve the absorption of calcium by the intestinal everted sac model, and that the absorption is similar to the CPP–Ca. In addition, egg whites are easy to obtain and the price is low, which makes EWP-Ca, an attractive new type of calcium supplement. The peptides obtained in related studies are mostly oligopeptides, consisting of 5–8 amino acid residues. However, the egg white peptides in this study are limited by the preparation process. The peptides obtained are mostly composed of about 10+ amino acid residues, making the peptide conformational easily affected by environmental factors, thereby decreasing the interaction between calcium ions and peptides. This may be the reason why the sterilization treatment can influence calcium-binding capacity. Subsequent research will be based on the perspective of industrial development, and it is possible to use a complex enzyme hydrolysis method to improve hydrolysis efficiency, reducing the influence of peptide conformational changes on the calcium binding. Future studies could also investigate how processes such as microencapsulation and Pickering emulsification immobilize the EWP-Ca conformation and improve thermal stability.


Listeria monocytogenes with Lactobacillus plantarum on the anti-Listeria activity of a hydrogen peroxide/peracetic acid based commercial disinfectant  when using conditions approaching the food industry environment. Nine strains of L. monocytogenes, including eight persistent strains collected from the meat industry and one laboratory control strain, were used in mono and in dual-species biofilms with a strain of L. plantarum. Biofilms were produced on stainless steel coupons (SSCs), at 11°C (low temperature) or at 25°C (control temperature), in TSB-YE (control rich medium) or in 1/10 diluted TSB-YE . The biofilm forming ability of the strains was evaluated by enumeration of viable cells, and the antibiofilm activity of P3 was assessed by the log reduction of viable cells on SSC. In both nutrient conditions and at low temperature, there was no significant difference (p > 0.05) between L. monocytogenes biofilm forming ability in mono- and in dual-species biofilms. In dual-species biofilms, L. monocytogenes was the dominant species. However, it was generally more susceptible to the lower concentration of P3 0.5% (v/v) than in pure culture biofilms. The presence of L. plantarum, although without significant interference in the number of viable cells of L. monocytogenes, enhanced the efficacy of the anti-Listeria activity of P3, since dual-species biofilms were easier to control. The results presented here reinforce the importance of the investigation into co-culture biofilms produced in food industry conditions, namely at low temperatures, when susceptibility to sanitizers is being assessed.The aim of this work was to investigate the effect of dual-species biofilms of  Listeria monocytogenes is an opportunistic foodborne pathogen responsible for listeriosis, a disease with high mortality rates  can adapt and grow under different environmental conditions . It is tironment . Lactoba animals . Many fa animals . Therefo animals .Staphylococcus aureus and Salmonella had a negative effect on the antimicrobial resistance of each microorganism to cetrimonium bromide, peracetic acid, and sodium hypochlorite, compared with the monospecies biofilms  treatment . Biofilms were produced in environmental conditions similar to those found in food processing plants after a poor cleaning procedure .The objective of this work was to evaluate the effect of L. monocytogenes and one strain of L. plantarum were used in this study. The strains of L. monocytogenes were characterized as previously described  and grown overnight at 37°C. Lactobacillus plantarum was streaked onto Man-Rogosa-Sharpe  and grown for 48 h at 37°C. Subsequently, isolated colonies were picked from the plates to prepare a work collection into the respective semi-solid medium [MRS was supplemented with CaCO3 (1 g L−1) in order to neutralize the acid produced and preserve the viability of the cultures  containing 15% (v/v) glycerol and stored at −80°C. cultures ]. After L. monocytogenes strains from the work collection were inoculated onto TSA-YE plates. After a 24 h period of incubation at 37°C, the inocula was prepared by suspending one isolated bacterial colony in 10 ml of TSB-YE or 1/10 diluted TSB-YE (approximately 107 CFU ml−1 confirmed through plating). Similarly, L. plantarum was inoculated onto MRS agar and incubated for 48 h at 37°C. After this period, the procedure for inocula preparation was the same as for L. monocytogenes strains. The use of 1/10 diluted TSB-YE compared with the control rich medium (TSB-YE) intended to mimic the situation of biofilm formation after a deficient cleaning procedure in the food processing industry.In order to prepare the inocula for producing pure culture biofilms, P3-Oxonia active  was used for the evaluation of anti-Listeria activity. The active compounds of P3 are hydrogen peroxide, acetic, and peracetic acid. According to European Standard EN 13697 , P3 was The procedure for biofilm formation was as described by ø = 3 mm). Thirty sterile glass beads and 1 ml of Ringer’s solution were then added to each well. The microplate was stirred in a Microplate vortex  for 1 min at maximum speed (position 10) in order to remove the biofilm from both sides of the coupons. From each well, 100 μl of the suspension was withdrawn in order to perform serial decimal dilutions to spot inoculate (25 μl) TSA-YE plates. Colony forming units (CFUs) were counted after overnight incubation at 37°C. The assays were replicated using four replicates: two biologically independent cultures on distinct days , each with two repetitions under identical conditions .A coupon was placed per well, and 1.5 ml of the respective inoculum was added. Controls with non-inoculated coupons were present in each P24 microplate. Incubation was performed at 25°C (room temperature) for 48 h or at 11°C (low temperature) for 7 days, in both cases without agitation. After this period, in order to remove planktonic cells, each coupon was rinsed through pippeting 1 ml of Ringer’s solution  on each side of the coupon, which was then placed in a new P24 microplate that contained a layer of 20 sterile glass beads  into the wells with the coupons. After 24 h at 11°C, L. monocytogenes inoculum was added (1 ml) to the wells with L. plantarum in order to produce dual-species biofilms. This was needed since simultaneous co-inoculation resulted in complete absence of detectable cells of L. plantarum in the biofilms. The assays also included wells for pure culture L. plantarum biofilms in which, after 24 h, instead of L. monocytogenes inoculum, 1 ml of fresh TSB-YE was added. A similar procedure was performed when the biofilms were cultured in 1/10 diluted TSB-YE. Incubation proceeded at 11°C for 7 days without agitation.The procedure was the same to the one described for L. monocytogenes CFU. Plates were incubated at 37°C for 48 h before counting the colonies. Lactobacillus plantarum was indirectly calculated by subtracting to the total CFU numbers in TSA-YE, the number of L. monocytogenes in PALCAM. Similarly to pure culture biofilms, two biological replicates with two technical replicates were always performed.After this period, biofilms were removed as described in the mono-species biofilm paragraph, except that suspensions from co-culture biofilms were additionally plated onto PALCAM , in order to count L. monocytogenes biofilms cells or dual-species biofilm cells, respectively. The CFU count assessment was made after 24 h and confirmed after 48 h of incubation at 37°C. Two biological replicates were performed with two technical replicates, each.After biofilm formation, each coupon was rinsed with 1 ml of Ringer’s solution on both sides, placed in a new P24 plate and exposed to 1 ml of P3 at the respective concentration  and for the respective exposure time (5 or 10 min). For calculating log reduction, a control exposed to sterile water was used. After the exposure period, each coupon was rinsed with 1 ml of Ringer’s solution, and then transferred to a new P24 with a 20-glass bead layer in the bottom. A set of 30 glass beads was put on the top of each coupon and 1 ml of Dey/Engley neutralizing broth solution  was added for an efficient neutralization contact period of 5 min. After this step, the plate was stirred in a Microplate vortex for 1 min at maximum speed. From each well, 100 μl was directly inoculated and the remaining suspension was decimal diluted for spread inoculating TSA-YE plates or TSA-YE and PALCAM plates, for −2) on the surfaces (SSC) exposed to (i) hard water (enumeration of viable cells) and (ii) to the disinfectant (antibiofilm activity of P3). If that reduction was observed with the lower P3 concentration and time exposure, no more treatments were carried out. Otherwise, treatments with higher concentration and/or exposure time were performed until the 4-log reduction was reached.The disinfectant treatment was considered effective when a 4-log CFU reduction was obtained . The red−2) and antibiofilm activity of P3 (log reduction) assays was checked for agreement to the normal distribution and for homogeneity of variance  by using the MiniTab17 software . When normality and homogeneity of variance were confirmed, one-way ANOVA with Tukey’s test was performed to calculate statistical differences between average values. When the data did not comply with ANOVA assumptions, the non-parametric Kruskal–Wallis median test was used. A principal component analysis (PCA) and a Cluster Analysis (CA) were performed in order to compare the susceptibility of the nine L. monocytogenes strains in mono- and in dual-species biofilms with L. plantarum following exposure to P3 0.5% (v/v) for 5 min (log reduction). Biofilms were produced in SSC, for 7 days at 11°C, and grown in TSB-YE (control rich medium) or in 1/10 diluted TSB-YE . The CA was made using the hierarchic clustering method and Euclidean distance was used as a measure of similarity or distance between strains. The software used was Statistica version 7.0 . For all tests, a probability greater than 95% (p < 0.05) was considered as significant.Data from biofilm formation on SSC ; and at low temperature  compared with the ambient control temperature (25°C for 48 h). The different periods of incubation were previously calculated in order that biofilms produced at both temperatures presented similar levels of L. monocytogenes CFU. P3 antibiofilm activity was tested in rising concentrations  of the disinfectant until the 4-log threshold was reached ], with the lower concentration of P3 [0.5% (v/v)], a general low susceptibility (<4-log CFU reduction) of the strains was observed . This efSince none of the nine isolates reached the 4-log reduction threshold in the four conditions tested, P3 concentration and the time of exposure were increased to 1 and 2% (v/v) for 10 min. With 1% (v/v) P3, all the strains, at least in one condition, reached the 4-log reduction threshold . HoweverL. monocytogenes in monoculture produced at low temperature were less susceptible to disinfectants, the assays with dual-species biofilms proceeded at 11°C, in 1/10 TSB-YE as well as in rich control medium TSB-YE. The results shown that in general, regardless the growth medium, L. monocytogenes was the dominant species in the dual-species biofilms with significantly higher numbers of viable cells (p < 0.05) (p > 0.05) between the average numbers of log CFU cm−2 of Listeria and the total biofilm population. Moreover, in both nutrient conditions, there was no significant difference (p > 0.05) between L. monocytogenes cell enumeration in mono- and in dual-species (As biofilms of  < 0.05) . There w-species .Listeria activity of the disinfectant was compared between mono- and dual-species biofilms with L. plantarum. LAB was never detected after the exposure of biofilms to any concentration of P3 tested suggesting that the eventual survivors would be outside the detection limit of the method. When grown in TSB-YE and exposed to 0.5% (v/v) P3, from the nine tested strains, six in mono-species  were below the 4-log reduction threshold (L. plantarum (L. plantarum (p < 0.05) (Biofilms were exposed to P3  and the hreshold . This eflantarum . In this < 0.05) . The con < 0.05) . In thes < 0.05) .L. monocytogenes strains based on their response to the lower concentration of P3 tested [0.5% (v/v)] was investigated. Data from log-reduction of mono and-dual species biofilms grown in both nutrient media, TSB-YE and 1/10 TSB-YE, at 11°C, were used for PCA. The initial four-dimensional space (four variables/conditions) were reduced to a plane F1F2 defined by the two first principal components. The projection of the nine strains in this plane, which accounted for 70.4% of the variability of the data, showed that the eight persistent strains were gathered in a major cluster. Strains S2-1, S1(S), S1(R), R6, and S10-1 with higher log reduction values when in mono species grown in 1/10 TSB-YE and in dual-species in TSB-YE; and strains P12, S2-2, and A7 with lower log reduction values in the same conditions. Strain EGD-e, although with lower log reduction values when in mono species grown in 1/10 TSB-YE and in dual-species in TSB-YE was well separated from the eight persistent strains (data not shown). Cluster analysis  for 10 min, the majority of the strains in mono- and dual-species biofilms reached the reference level of reduction (L. monocytogenes biofilms to disinfectant treatment. L. monocytogenes and L. plantarum showed enhanced resistance to BAC and peracetic acid. agr) locus of L. monocytogenes is important in the initial adhesion phase of biofilm production by L. monocytogenes, and the maximum expression of agr locus was observed at 37°C, whereas expression was the lowest at 10°C  on the results obtained. Consequently, special care should be taken in generalizing biofilm results since the particular environment in which they are formed may affect biofilm susceptibility. The results presented here indicate that proteome . It is pormation . Longer Lactobacilli have been considered as GRAS , which identifies a microorganism or microbial derivatives as safe for use in the food industry  after a deficient cleaning procedure (nutrient deprivation). Results showed that although L. monocytogenes was the dominant species in dual-species biofilms with L. plantarum, it was generally more susceptible to low P3 concentration [0.5% (v/v)] than in mono-species. While dual-species were generally easier to control, biofilms produced under conditions approaching the real food industry environment may be more difficult to eradicate, resulting in the presence of persistent strains in food facilities and/or food products. These results will complement other ongoing research into the persistent L. monocytogenes strains used. More researches under conditions closer to the food industry environment are welcomed, for instance refrigeration temperatures, multi-species biofilms, different types of surfaces, and new disinfectants including natural active compounds.Even though there is much information available on single species biofilms, mixed species biofilms are more probable to occur in the food processing areas. In the present study, the effect of dual-species biofilms of The original contributions presented in the study are included in the article/LBo performed some of the assays and prepared the manuscript. VM executed the majority of the experiments. IB participated in the experiments. LBr projected the study, assessed, and interpreted the results. LBo and LBr wrote the manuscript. JM-S and LBo assessed and interpreted the results. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background and Objectives: High-sensitivity cardiac troponin I (hs-TnI) is an important indicator of acute myocardial infarction (AMI) among patients presenting with chest discomfort at the emergency department (ED). We aimed to determine a reliable hs-TnI cut-off by comparing various values for a baseline single measurement and an algorithmic approach. Materials and Methods: We retrospectively reviewed the hs-TnI values of patients who presented to our ED with chest discomfort between June 2019 and June 2020. We evaluated the diagnostic accuracy of AMI with the Beckman Coulter Access hs-TnI assay by comparing the 99th percentile upper reference limits (URLs) based on the manufacturer’s claims, the newly designated URLs in the Korean population, and an algorithmic approach. Results: A total of 1296 patients who underwent hs-TnI testing in the ED were reviewed and 155 (12.0%) were diagnosed with AMI. With a single measurement, a baseline hs-TnI cut-off of 18.4 ng/L showed the best performance for the whole population with a sensitivity of 78.7%, specificity of 95.7%, negative predictive value (NPV) of 97.1%, and positive predictive value (PPV) of 71.3%. An algorithm using baseline and 2–3 h hs-TnI values showed an 100% sensitivity, 97.7% specificity, an NPV of 100%, and a PPV of 90.1%. This algorithm used a cut-off of <4 ng/L for a single measurement 3 h after symptom onset or an initial level of <5 ng/L and a change of <5 ng/L to rule a patient out, and a cut-off of ≥50 ng/L for a single measurement or a change of ≥20 ng/L to rule a patient in. Conclusions: The algorithmic approach using serial measurements could help differentiate AMI patients from patients who could be safely discharged from the ED, ensuring that patients were triaged accurately and did not undergo unnecessary testing. The cut-off values from previous studies in different countries were effective in the Korean population. Chest pain is a common symptom observed in clinical practice and a symptom that suggests acute myocardial infarction (AMI). Approximately 10% of all patients who visit the emergency department (ED) present with symptoms suggestive of AMI; however, 75–85% of them are found to have non-coronary diseases ,3. ThereCardiac troponin (cTn) is a key indicator for early diagnosis, especially in non-ST segment elevation myocardial infarction (NSTEMI) without remarkable changes in the electrocardiogram (ECG) . The impBeckman Coulter  recently released a new version of the high-sensitivity troponin I (hs-TnI) assay called the Access hs-TnI assay, which was approved in 2018. In a previous study, we found that the Access hs-TnI assay met the performance criteria of hs-cTn assays, and we also reported that the 99th percentile upper reference limits (URLs) of the Korean population were lower than the manufacturer’s claims and differed according to sex and age ,7.The international guidelines recommend using the 99th percentile URL calculated using healthy subjects as a cut-off to diagnose myocardial injury . HoweverThe purpose of this study was to evaluate the possibility of a rapid AMI diagnosis and safe discharge using the Beckman Coulter Access hs-TnI assay in Korean patients who visited the ED. We aimed to determine a safe and reliable diagnostic criteria by comparing the diagnostic accuracy using the 99th URLs based on the manufacturer’s claims, the newly designated URLs in Korean populations, and the algorithms provided by other researchers ,15.This study was a single-center retrospective observational study of patients who visited the ED of the Sanggye Paik Hospital between June 2019 and June 2020. The inclusion criteria were: (1) aged > 20 years; (2) presence of chest pain or equivalent ischemic symptoms (including epigastric or left upper quadrant pain or discomfort or pressure); and (3) measurement of hs-TnI levels at ED presentation. The exclusion criteria  were: (1) clear alternative cause for the suspected symptoms other than acute coronary syndrome (including trauma); (2) inter-hospital transfer for revascularization after initial blood tests; (3) discharge against medical advice without adequate work-up; (4) cardiac arrest or in-hospital death at the ED before adequate work-up; (5) history of coronary revascularization within 3 weeks; and (6) diagnosis of ST segment elevation myocardial infarction (STEMI) on presentation. The protocol was approved by the Institutional Review Board of Sanggye Paik Hospital, Seoul, Korea (SPIRB-2019-05-014) and the requirement for informed consent was waived due to the retrospective study design.Patient demographic information, risk factors, previous history of coronary artery disease, presenting symptoms, and time between symptom onset and ED visit, were extracted electronically from medical records. In addition, examination findings such as vital signs, ECG, laboratory results, coronary angiography, echocardiography, and radiologic findings, such as enhanced chest computed tomography (CT), thoracic aorta CT angiography, and pulmonary CT angiography, were collected. Final diagnoses were adjudicated retrospectively by a cardiologist and a physician in the ED by reviewing all available medical records pertaining to the patient from the time of ED presentation to their most recent follow-up of at least 6 months. AMI, an outcome indicator, was defined as recommended by the 4th universal definition guidelines . MyocardThe hs-TnI values were measured with the Access hs-TnI assay on the UniCel DxI800 analyzer platform (Beckman Coulter) in the central laboratory of our hospital within an hour after blood was drawn. The hs-TnI results analyzed in this study included the baseline hs-TnI at the ER visit (0 h) and a second hs-TnI measurement performed two to three hours after the initial blood test (2–3 h). On the package insert of the Access hs-TnI assay, the manufacturer described a limit of detection of 2.3 ng/L and limit of quantification at 10% coefficient of variation (CV) of 5.6 ng/L. The manufacturer suggested an overall 99th percentile URL of 17.5 ng/L  with a corresponding CV of <10%. In our previous study, we noticed that our population showed a lower 99th percentile URL compared to the insert . We also noticed that the 99th percentile URLs of hs-TnI were higher in men  and in the age group ≥50 years . In this study, we compared the clinical performance of the 99th percentile URLs suggested by the manufacturer and calculated in our previous study. We used the concept of the current hs-cTn 0/2–3 h algorithms suggested by groups in Europe, Australia, and United States ,16. ThisFor the continuous variables, the median and interquartile range (IQR) were calculated, and a Student’s t-test or Mann–Whitney test was performed. For categorical variables, the frequency and percentage were calculated and a Chi square test or Fisher’s exact test was performed.The sensitivity, specificity, PPV, and NPV were evaluated for the baseline hs-TnI values using the various 99th percentile URLs. Area under the curve (AUC) from a receiver operating characteristic (ROC) curve was calculated to compare the clinical performances of the different baseline hs-TnI cut-off values. Youden’s indexes (sensitivity + specificity − 1) were calculated to select the optimal cut-offs for initial hs-TnI values. p < 0.05.To evaluate the safety of the algorithmic approach, the sensitivity and NPV were calculated in the Rule-out group, and the specificity and PPV were calculated in the Rule-in group for accuracy evaluation. In addition, the percentage of patients classified in the Rule-out group or the Rule-in group was calculated to evaluate the efficacy. Statistical analyses were performed using Analyse-it for Microsoft Excel 5.40.2  and SPSS 18.0 , and the significance level was set to p = 0.004). The frequencies of the risk factors such as diabetes, hypertension, and heart failure were higher in the AMI group than in the non-AMI group, although they did not reach the statistical significance values. The frequencies of previous coronary artery diseases  and lower estimated glomerular filtration rates  were significantly higher in the AMI patients than in non-AMI patients.A total of 1410 patients visited the ED with chest pain or the equivalent symptoms and underwent an hs-TnI test at presentation. Forty patients were excluded due to inter-hospital transfer for revascularization ; recent coronary revascularization ; discharge against medical advice without adequate work-up ; cardiac arrest in ED  or in-hospital death within 24 h due to medical illness including cardiogenic shock, malignancies, or acute cerebrovascular events ; and multiple trauma including traffic accidents, chest contusion, and fall-down accidents . Among 1370 patients, 74 patients who were diagnosed with STEMI were excluded from further analysis . A total of 1296 patients were analyzed for the diagnostic performance of hs-TnI at the ED . The basA total of 155 (12.0%) patients were diagnosed with AMI, including 107 (8.3%) patients with Type 1 MI and 48 (3.7%) with Type 2 MI. Other patients were grouped into non-MI , including unstable angina , chronic coronary syndromes , non-coronary cardiac diseases , and non-cardiac diseases . p < 0.0001). The baseline Access hs-TnI levels were calculated according to their final adjudicated diagnosis, and the medians and IQRs were as follows: 96.5 ng/L (22.8–747.2) in Type 1 MI; 38.4 ng/L (13.3–124.8) in Type 2 MI; 5.0 (3.1–8.0) in unstable angina; 3.0 (2.3–5.4) in chronic coronary syndromes; 5.1 (3.0–9.8) in non-coronary cardiac diseases; and 3.0 (2.3–5.0) in non-cardiac diseases A. The Acp < 0.0001). The delta-TnI levels were calculated according to the final adjudicated diagnosis, and the medians and IQRs were as follows: 144.3 ng/L (36.2–648.5) in Type 1 MI; 25.4 ng/L (4.0–134.7) in Type 2 MI; 1.0 (0.2–1.7) in unstable angina; 0.3 (0.0–1.0) in chronic coronary syndrome; 0.7 (0.0–1.0) in non-coronary cardiac diseases; and 0.3 (0.0–1.0) in non-cardiac diseases (Second follow-up hs-TnI levels were measured in 589 patients (45.4%) between 2–3 h after the first blood test (median 160 min). Delta hs-TnI levels were significantly higher in the AMI group  than in the non-AMI group  and NPVs (p > 0.05) but consistently poor specificities and PPVs (p < 0.001), both in the total population and all subgroups  [The diagnostic performance of baseline Access hs-TnI levels for the diagnosis of AMI was measured using the AUC, and the results were as follows: 0.934  in total; 0.921  in men; 0.974  in women; 0.930 (0.904–0.955) in those aged ≥50 years; and 0.928 (0.860–0.997) in those aged <50 years . The priIn the hs-TnI Access 0/2–3 h algorithm, 912 out of 1296 patients (70.4%) could be analyzed. There were 384 patients excluded from algorithm analysis, who could not be ruled in or ruled out using the initial hs-TnI values and had no adequate second hs-TnI measurement 2–3 h after the first blood test. The selected algorithm used a cut-off of <4 ng/L for a single measurement >3 h after the onset of symptom or an initial level of <5 ng/L and a change of <5 ng/L to rule a patient out, and a cut-off of ≥50 ng/L for a single measurement or a change of ≥20 ng/L to rule a patient in . Among tAmong the patients in the Rule-out group and Observe group, 55.1% (425/771) were followed up for longer than one month . The 30-An internal validation was performed to confirm the reproducibility of the algorithmic approach. The total patients (N = 1296) were divided into derivation (60%) and validation (40%) cohorts according to the chronological order of ED visits. After excluding the patients who could not be classified into Rule-in or Rule-out with initial hs-TnI and who did not have an adequate second hs-TnI measurement, 518 and 394 patients, respectively, were included in the derivation and validation cohorts . The difThe recent improvements in the analytical performance of hs-cTn assays enabled the detection of low levels of cTn and subtle changes in cTn and these assays played an important role in the clinical judgement for AMI in the ED. This study was performed to evaluate the diagnostic accuracy and clinical usefulness of the Access hs-TnI assay as an early screening tool for the detection of AMI. p = 0.1133), and the lower eGFR <60 mL/min/1.73 m2   . The patIn the previous study, we set the 99th percentile URL in our population according to international guidelines ,8. In thThe prime cut-off values calculated from the ROC curves were not significantly different in men 18.8 ng/L) and women 17.5 ng/L) with proximities to the manufacturer’s 99th percentile URL . However, the best cut-off values from the ROC curves were significantly lower in patients <50 years (6.9 ng/L) compared to patients ≥50 years (16.8 ng/L). This finding was the same when we divided the groups with a cut-off of 65 years . The previous guidelines emphasized the sex-specific 95th percentile URLs, but these data suggested that it may be necessary to consider URLs based on age groups .8 ng/L a. This wa.5 ng/L wTo overcome the shortcomings of the diagnostic performance with a single cut-off, many algorithmic approaches were proposed. We were also able to confirm that the algorithmic approach resulted in a higher accuracy  and more reliable predictabilities  than the single measurements of any of the cut-offs  or Rule-out (50.7%) groups without the need for the second cTn test, so this algorithm was useful for establishing a rapid decision-making strategy.Nevertheless, when applying the 0/2–3 h algorithm using hs-TnI levels in clinical practice, the following should be noted. In this study, 176 (19.3%) patients were in the Observe group, and 18 (10.2%) of these patients were diagnosed with AMI. Separating the Observe group enabled the selection of patients who needed further work-ups and avoided risky discharge, even if hospitalization and rapid revascularization were not required. Other studies reported that the Observe group patients had lower 30-day mortality rates, but similar 1-year mortality rates, compared to the Rule-in group patients . TherefoIn particular, it is known that a single measurement requires the careful consideration of the time elapsed since symptom onset ,21. In tFurthermore, it should be noted that 90.1% of the patients assigned to the Rule-in group were patients with AMI, and they required urgent percutaneous coronary intervention. In addition, even if type 1 MI was not diagnosed in the Rule-in group, those patients presented with unstable conditions requiring hospitalization, such as heart failure, myocarditis, unstable angina, acute cholecystitis, acute pyelonephritis, and pulmonary embolism. Therefore, in order to differentiate between these diseases, the coronary angiography for diagnosis and treatment could be considered for patients in the Rule-in group.This study had several limitations. First, as a retrospective study, this study included patients who underwent hs-TnI on ED visits, and some patients who did not undergo a second hs-TnI test within 2–3 h were excluded. If the hs-TnI test was performed late or omitted based on the judgment of the medical staff, the patient was excluded from this algorithmic analysis. This could lead to selection bias and the omission of some cases of AMI. Second, it was difficult to guarantee the long-term safety of the Rule-out or Observe group in the 0/2–3 h algorithm because 44.9% (346/771) of patients did not visit our outpatient clinic after one month and a long-term follow-up was not performed in this study. Further prospective studies are required to compensate for these weaknesses and to verify the outcomes. Third, since the patients were recruited from a single regional ED, the local characteristics of the institute could impact the results. A multi-center prospective study is recommended to verify the accuracy and usefulness of the hs-TnI test and this algorithmic approach. Fourth, it is desirable to validate the analysis performance of the algorithm in each gender and in patients with renal impairments. However, in this study, subgroup analysis was not possible as the number of AMI was small in women  and the low eGFR group . In the future, we plan to conduct additional subgroup analyses through a prospective study with more patients. Nevertheless, this study was meaningful as it aimed to assess the clinical performances of Access hs-TnI in Koreans, whereas most of the published data were from one multinational study group or from Western countries. This study was the first to attempt an algorithmic approach for the diagnosis of AMI using the Access hs-TnI assay in an Asian population, involving the independent evaluation of the 99th URLs within the same institution.In summary, the algorithmic approach, rather than the use of single hs-TnI values, was a more efficient measurement for the early diagnosis of AMI and safe discharge from the ED. We found that the cut-off values used in the algorithmic approaches from previous studies in different countries were effective in the Korean population.


Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. The cyclomarins are also very potent inhibitors of Plasmodium falciparum. Biosynthetically the cyclopeptides are obtained via a heptamodular nonribosomal peptide synthetase (NRPS) that directly incorporates some of the nonproteinogenic amino acids. A wide range of derivatives can be obtained by fermentation, while bioengineering also allows the mutasynthesis of derivatives, especially cyclomarins. Other derivatives are accessible by semisynthesis or total syntheses, reported for both natural product classes. The anti-tuberculosis (anti-TB) activity results from the binding of the peptides to the N-terminal domain (NTD) of the bacterial protease-associated unfoldase ClpC1, causing cell death by the uncontrolled proteolytic activity of this enzyme. Diadenosine triphosphate hydrolase (PfAp3Aase) was found to be the active target of the cyclomarins in Plasmodia. SAR studies with natural and synthetic derivatives on ilamycins/rufomycins and cyclomarins indicate which parts of the molecules can be simplified or otherwise modified without losing activity for either target. This review examines all aspects of the research conducted in the syntheses of these interesting cyclopeptides.Ilamycins/rufomycins and cyclomarins are marine cycloheptapeptides containing unusual amino acids. Produced by  Marine organisms produce a wealth of natural products, creating a universe of fascinating new chemical structures ,2. TheseMalaria is one of the most common tropical diseases, with more than 200 million infections and 600,000 deaths annually worldwide . In addiStreptomycetes from soil samples found on Japanese islands. Takita et al. observed that the culture filtrate of a new strain, Streptomyces insulates (A-165-Z1), later renamed Streptomyces islandicus, inhibited the growth of Mycobacterium 607 and Mycobacterium phlei. They isolated two antibiotics and named them ilamycin A and B (IlaA and IlaB) ,3627,36]d to AHA . An N-prarginine . AdditioN-prenyl side chain by a P450 epoxidase occurs post-NRPS assembly. Gene inactivation of the prenyltransferase eliminated the production of cyclomarin, indicating that p-Trp is the initiator of the peptide assembly line and that the unmodified p-Trp is not a good substrate for the first acceptor domain ,3534,35]Streptomyces sp. CNB-982, together with CymD that was missing the N-methyl group on the δ-hydroxyleucine ②. It is clear that δ-hydroxylation by P450 leucine hydroxylase occurs faster than N-methylation, and tryptophan hydroxylation is essential for prolonging the peptide chain. If this hydroxylation does not occur properly, the incompletely processed dipeptide is cleaved from the NRPS due to ineffective processing by the next module. This reaction is a unique example in which a megasynthetase can produce two different natural products of different sizes simply by triggering the level of β-oxidation on the priming Trp unit. Notably, cyclomarazines do not show significant biological activity.CymA and CymB were isolated from the O-methylation . A. A32]. AThe interesting biological properties and unusual building blocks of marine cycloheptapeptides sparked the interest of synthetic chemists, and the syntheses of several different amino acids and fragments have been reported in a recent review . Therefo1 and 2) that were linked between ① and ⑦ to the macrocyclic lactam (1 (①–③) was prepared in five steps from tryptophan, while the upper left tetrapeptide 2 (④–⑦) required 13 steps from glutamic acid. Final oxidation of the δ-hydroxyleucine ⑤ resulted in the described ilamycins.To date, only one synthetic route has been described for ilamycins E1 and F by Guo and Ye et al. . In theic lactam . The low1 was rather straightforward , and the Boc-amide was selectively N-methylated to 8 with NaH/MeI. The free carboxylic acid 8 was converted into the corresponding benzyl ester. TMSOTf/NEt3 was used for selective cleavage of the N-Boc-protecting group without affecting the OTBDPS group. The free amine could be coupled with Fmoc-protected alanine, and the C-terminal benzyl ester was cleaved by catalytic hydrogenation to provide the free acid 9.For the larger fragment tected 8  accordinr moiety ,42. For 10 .,55.54,55N-terminus failed. Under the basic conditions for Fmoc-deprotection, spontaneous cyclization to the corresponding diketopiperazine occurred, comparable to the previously discussed biosynthetic side reaction, which resulted in the formation of the cyclomarazines. The ultimately successful route was the cyclization between the unsaturated amino acid ⑦ and the C-terminal N-methylleucine ⑥. The linear heptapeptide was obtained via a [4+3]-coupling strategy. An allyl ester was used as the C-terminal protecting group to avoid the basic reaction conditions required for the saponification of the C-terminal ester, which caused problems in previous cyclization attempts.After the desired building blocks were created, the synthesis of cyclomarin C and especially the best position for macrocyclization was investigated  47,56].,56.47,5639 and 40 were synthesized using classical peptide coupling reactions and a combination of Boc- and Fmoc-protecting groups 2Pyr as chiral ligand, but the ee did not exceed 80 % [53. The iodides 52 and 53 were next converted into organometallic reagents and reacted with a protected serinal. While the corresponding Grignard reagents provided only moderate yields and selectivities, zinc reagents were found to be superior. According to Knochel et al. [52 was presumably converted into the indole–zinc–magnesium complex 54a, which was reacted with freshly prepared protected serinal to give the desired syn-configured 55a as a single diastereomer. In the case of the epoxyindole 53, a slightly different protocol was used. To avoid side reactions during the metalation step, 53 was lithiated at −78 °C with n-BuLi and transmetallated with ZnBr2 [54b was directly reacted with the aldehyde to create 55b. According to NMR and HPLC, only two diastereomers (ratio 9:1) could be detected following the Sharpless dihydroxylation step. Obviously, the carbonyl addition also here was highly stereoselective. Next, the secondary OH-functionality was TBS-protected under the assumption that a primary OTBS-group could be removed selectively [56a in high yield, while all other methods failed and resulted in the decomposition of 55a. Interestingly, no complete conversion was obtained for 55b, but the silyl ether 56b was obtained as a single stereoisomer. Obviously, the undesirable diastereomer did not undergo silylation. Next, the primary silyl protecting group was removed using NH4F in MeOH [57 had to be oxidized to the desired carboxylic acids 59, which were found to be highly sensitive and not very stable. By far, the best results were obtained using a two-step protocol beginning with a Parikh–Doering oxidation [58 with N-iodosuccinimide in MeOH [59 on demand.The second synthesis of cyclomarin C and the first for cyclomarin A were reported in 2016 by Barbie and Kazmaier . Both na indoles . Using ia et al. , 52 was eed 80 % . Subsequl et al. ,64, 52 wth ZnBr2 ,64. The ectively . Notably in MeOH . The frexidation . The als in MeOH . These aS)-Roche ester, which was O-silylated to 60 (61 [62 obtained was subjected to asymmetric hydrogenation [R)-MonoPhos as a chiral ligand [63 and N-methylation yielded the desired building block 64.A straightforward protocol was developed for the protected δ-hydroxyleucine ②, starting with the commercially available -serine 65 was reduced to the corresponding aldehyde, which was subjected to a chelate-controlled aryl-metal addition. The addition of phenylmagnesium bromide provided an acceptable yield but only moderate diastereoselectivity (7:3). In contrast, in the presence of titanium salts, the desired coupling product 66 could be obtained as a single diastereomer in enantiomerically pure form. Further standard transformations yielded building block 32.The third unusual amino acid, β-methoxyphenylalanine ④, could be obtained similarly to the tryptophan building block ① . Protect67 -selective olefination [73 could be obtained almost epimerization-free. Saponification and change in the N-protecting group provided amino acid 74.For the synthesis of the fourth, the unsaturated amino acid ⑦, also a chelate enolate Claisen rearrangement, was used starting with chiral ester 70  [74,75,7N-methylleucine 75 (⑥) to cyclize the linear heptapeptide at the same position as achieved by Yao et al.  but also the malaria parasite Plasmodium falciparum [83, which was linked to sepharose beads and processed by affinity chromatography to identify the target. Interestingly, 83 inhibits the growth of Mtb similarly to cyclomarin A [N-prenyl side chain is not responsible for the biological activity, at least against Mtb. In contrast, replacing the terminal OH-group of the δ-hydroxyleucine ② by an N-methylanilin (84) caused a significant drop (>200 fold) in activity [Researchers at Novartis observed that cyclomarin A might be a good candidate for drug development since it kills not only lciparum . Thus, clciparum . Ring-opomarin A . Obvious77, which was very active against Mtb, demonstrated little activity against Plasmodium falciparum. Therefore, the Novartis researchers also synthesized acetylenic amine 85 for target fishing [86 with a missing tryptophan side chain was used. This compound was obtained by retro aldol reaction.Notably, the amine  fishing . This coN-Isopropyltryptophan was obtained via Negishi coupling of 3-iodo-N-isopropylindole with protected zincated iodoalanine [Retro aldol reaction of the β-hydroxytryptophan seems to be a serious stability issue, also during synthesis. This building block undergoes the discussed side reaction proceeding under slightly basic conditions. Under acidic conditions, water is rapidly eliminated, resulting in the formation of the α,β-unsaturated dehydrotryptophan derivative. To avoid these problems, Kazmaier et al. synthesized a series of cyclomarin derivatives containing non-hydroxylated tryptophans (desoxycyclomarins), e.g., the building blocks found in ilamycins/rufomycin ,86. N-Isoalanine . Severaloalanine . Other dStreptomycetes in 1962 as new antibiotics, active against acid-fast bacteria, especially Mycobacteria. Ilamycin A was reported to inhibit Mycobacterium 607 at 0.5 μg/mL, while ilamycin B was less active (3 μg/mL). The rufomycins were reported to be highly active against Mycobacterium smegmatis  and Mycobacterium tuberculosis , also strains resistant to other antibiotics such as streptomycin (SM), neomycin (NM), kanamycin (KM), and isonicotinic acid hydrazide  [Both the ilamycins ,15 and r0 mg/kg) .Streptomyces atratus ZH16 ΔilaR. The analogs demonstrated a slightly different oxidation pattern compared to the previously isolated ilamycins [Mycobacterium tuberculosis, while the most active examples thus far have been ilamycin E and J  from a 200 L scale culture of mutant lamycins ,28. Most E and J , both moBased on the bioassay data, some structure-activity relationships became evident. Cyclized compounds such as IlaE and IlaJ demonstrated greater activity than open-chain leucine derivatives such as IlaB, IlaD, or IlaF . OxidatiStreptomyces atratus strain MJM3502 [M. tuber. HR37v) similar to rifampicin (0.03 μM). Ruf I maintained activity against monoresistant, MDR, and XDR strains, indicating that the Rufs most likely have a different molecular target than current drugs used in TB therapy. Additionally, Ruf I retained its activity against strains from the five global M. tuberculosis clades representative of clinical TB disease worldwide [M. abscessus, one of the more difficult-to-treat mycobacteria [In 2020, Pauli et al. isolated eight new rufomycins (rufNBZ1-NBZ8) together with five already known derivatives from the  MJM3502 . Analogoorldwide . Especiabacteria .M. tuberculosis [M. tuberculosis. In this species, the strictly regulated ClpC1 associates with the proteolytic domains, ClpP1 and ClpP2, and together they are responsible for waste protein degradation within the cell [N-terminal helical domain (NTD) of ClpC1 and Ruf I reveals distinct differences from the corresponding ClpC1-NTD-cyclomarin A structure. Surprisingly, the complex structure shows that the epoxide moiety of Ruf I is opened and covalently bound to ClpC1-NTD via the sulfur atom of a methionine in the binding pocket [Detailed studies indicated that the ilamycins/rufomycins inhibit ClpC1, an enzyme currently not targeted for TB treatment. ClpC1 is the ATP-dependent homolog of the ClpC class of chaperone proteins present in rculosis  and is hthe cell . Withoutthe cell . In thisthe cell . The X-rg pocket ,93.Although the anti-tubercular activity is the most prominent effect of ilamycins/rufomycins, certain derivatives show some anti-cancer activities, while others do not. IlaC (Ruf I) was found to be preferentially cytotoxic towards triple-negative breast cancer (TNBC) cell lines, the most aggressive subtype of breast cancer with poor prognoses. IlaC can induce Bax/Bcl-2-related caspase-dependent apoptosis and inhibit migration and invasion in TNBC by suppressing IL-6-induced STAT3 phosphorylation . IlaE, bPlasmodium falciparum strains (IC50 0.25 μM) as well as anti-TB activity (MIC: 0.1 μM) [Candida albicans (IC50 > 50 μM). Schmitt et al. at Novartis investigated the mode of action in detail [83  showed anti-inflammatory activity in vivo and in vitro . Lateron 0.1 μM) . No signn detail . CymA watail [83  demonstrMycobacterium species is close to 95%, but the NTD of mycobacterial ClpC1 is 100% conserved. This phenomenon explains why all tested mycobacteria were found to be sensitive to CymA. Based on the structure of the complex, several mutations were engineered into ClpC1, which showed reduced CymA binding in vitro. The ClpC1 mutants were overexpressed in mycobacteria and two showed resistance to CymA, providing clear evidence that ClpC1 is the target of CymA.The exact binding of CymA toward the NTD of ClpC1 was determined by high-resolution co-crystal structure analysis . The oveUsing NMR and small-angle X-ray scattering, Schanda and Fraga et al. demonstrated that arginine-phosphate binding to the ClpC1 NTD induces millisecond dynamics . CyclomaPlasmodium falciparum, and its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), was identified by chemical proteomics [50: 0.004 μM) but not of the closest human homolog hFHIT (IC50 > 10 μM). Co-crystallization experiments demonstrated a unique inhibitor binding mode. One molecule of CymA binds a dimeric PfAp3Aase and prevents the formation of the enzyme-substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. Thus, CymA is a rare example of a natural product with two distinct and specific modes of action.However, anti-TB activity is not the only interesting feature of the cyclomarins. Schmitt et al. showed that CymA is a potent growth inhibitor of oteomics . CymA isUnfortunately, CymA as a natural product lacks satisfactory pharmacokinetic properties, making it challenging for optimization into an  bioavailable drug. Therefore, Kazmaier et al. tried to simplify the complex structure of the cyclomarins without losing significant biological activity. Because the β-hydroxytryptophan unit ① is the most critical building block, they removed the hydroxy functionality completely, and the desoxycyclomarins obtained were further modified on the prenyl side chain of ①, the hydroxylated leucine ②, the β-methoxyphenylalanine ④ and the unsaturated amino acid ⑦ ,73,85,861 were well-tolerated (series 87). Reduction to an isopropyl group (87c) provides an especially promising simplification retaining antimycobacterial activity. In general, manipulations at this position do not result in dramatic effects on potency measured against Mtb and Pfalcp. Interestingly, the methyl group in 87d is an appropriate balance between reducing synthetic complexity and loss of activity.Certain modifications at R2 were consistent with the data obtained by X-ray structure analysis [Pfalcp, where this residue is completely buried between the target and the ligand scaffold, large changes are not accepted. However, it is important that removing the terminal methyl group in the cis position of the γ,δ-unsaturated side chain led to an equivalent or even slightly improved activity. Further simplifications, however, are not advisable, as they lead to dramatic activity losses, which are seen in the comparison of 87a and 88a. In contrast, the anti-TB activity was not influenced.Results obtained for the modifications at Ranalysis . In the 3 led to a decrease in potency against both pathogens (89). This result was indeed expected for the Mtb target ClpC1-NTD [Pfalcp.Removal of the methyl group at RlpC1-NTD . This gr4 on β-methoxyphenylalanine ④ were also well tolerated (90). The amino- and azido-derivatives 90a and 90b were equipotent to cyclomarin C, and the nitro compound 90c was even twice as active. Only in the case of bromo derivative 90d was a significant drop in activity observed.Modifications of RStreptomyces sp. and show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis. No significant activity has been observed towards other Gram-positive and Gram-negative bacteria or fungi.The ilamycins/rufomycins and the cyclomarins are highly interesting marine cycloheptapeptides characterized by their incorporation of unusual amino acids. The natural products are produced by Plasmodium falciparum, the organism that causes malaria. Biosynthetically, the cyclopeptides are obtained via a heptamodular NRPS that directly incorporates some of the nonproteinogenic amino acids, while oxidations at certain positions allow the compounds to proceed to protein-bound biosynthetic intermediates. Cyclized ilamycins/rufomycins are obtained by oxidative post-NRPS cyclization of leucine ⑦, the last introduced amino acid in the biosynthesis. A wide range of derivatives can be obtained by fermentation, while bioengineering also allows the mutasynthesis of derivatives, especially cyclomarins. Other derivatives are accessible by semisynthesis or total syntheses, reported for both natural product classes. Some of these derivatives were used to identify the biological targets of these peptides. The anti-TB activity results from the binding of the peptides to the N-terminal domain (NTD) of the protease ClpC1, causing cell death by the uncontrolled proteolytic activity of associated enzymes.The cyclomarins are also very potent inhibitors of Diadenosine triphosphate hydrolase (PfAp3Aase) was found to be the active target of the cyclomarins in Plasmodia, and this enzyme might be a good candidate for the treatment of malaria. SAR studies of natural and synthetic derivatives on the ilamycins/rufomycins and cyclomarins indicate which parts of the molecules can be simplified/modified without losing activity towards either target.


HIV stigma is a common barrier to HIV prevention, testing, and treatment adherence, especially for low- and middle-income countries such as Myanmar. However, there was no validated Myanmar version of a stigma scale.Therefore, we adapted the English version of the 40-item Berger’s HIV stigma scale and the 7-item Indian HIV stigma scale into a 47-item Myanmar HIV stigma scale and then evaluated the scale’s psychometric properties.From January 2020 to May 2020, using random sampling methods, 216 eligible Myanmar people living with HIV/AIDS (PLWHA) were contacted from a closed Facebook group that included more than 10,000 PLWHA. A sample of 156 Myanmar PLWHA completed the online self-reported survey.p < 0.01). In Rasch analysis, the scale achieved person reliability of 3.40 and 1.53 and a separation index of 0.92 and 0.70. The infit and outfit mean squares for each item ranged from 0.68 to 1.40. No differential item functioning across gender or educational level was found.A six-factor structure for the scale was determined through exploratory factor analysis, explaining 68.23% of the total variance. After deleting 12 items, the 35-item HIV stigma scale achieved Cronbach ‘s α of 0.72 to 0.95. Construct validity of the scale was demonstrated by significant association with self-reported depression and social support levels (r = 0.60, and − 0.77, The psychometric properties of the 35-item Myanmar version of the HIV stigma scale support it as a measure of stigma among PLWHA in Myanmar. This instrument could help healthcare providers to better understand how stigma operates in PLWHA and to develop tailored stigma-reduction interventions in Myanmar.The online version contains supplementary material available at 10.1186/s12889-021-11685-w. HIV has become one of the major public health challenges contributing to high disease burden globally, especially for low- and middle-income countries such as Myanmar . After THIV stigma—which refers to prejudice against, discrimination toward, and discrediting PLWHA and the groups or communities with which PLWHA associate—is a common barrier to HIV prevention, testing, and treatment adherence , 7. HIV Currently, there are limited social support systems in Myanmar for PLWHA. As a result of their serostatus, many PLWHA encounter stigma and discrimination from their family, community, and healthcare systems , 12, 14.Most of the stigma scales used in recent studies in Myanmar were widely used in Western countries , 18, andHIV stigma can be enacted, anticipated, or internalized, and it is important that an instrument can identify and differentiate these stigma mechanisms. With such information, researchers could design a culturally sensitive intervention to decrease HIV stigma . SeveralStigma is related to a specific context of culture and power , 20. TakMyanmar, which lies on the Southeast Asian mainland bordered by Bangladesh, China, India, Laos, and Thailand, has a rich history influenced by British expansionism in the 19th and early 20th centuries . EthnicaTo understand the mechanisms and status quo of HIV stigma experienced by PLWHA in Myanmar, the current study’s aim was to (1) describe a culturally appropriate scale to measure their HIV stigma, adapted from Berger’s HIV stigma scale  and someCOnsensus-based Standards for the selection of health status Measurement Instruments) checklist [This cross-sectional descriptive study was approved by the relevant institutional review boards and was conducted in Myanmar from January 2020 to May 2020. We culturally adapted the Berger and Indian HIV stigma scales to create the Myanmar version of the HIV stigma scale and examined the psychometric properties of the scale, which were adherent to COSMIN  system.We adapted the two HIV stigma scales to measure the stigma experienced by PLWHA in Myanmar in the following four stages:To create the 47-item HIV stigma scale in Myanmar (HIVSS-M-1), we adapted the 40-item Berger’s HIV stigma scale   completed the REDCap survey. After the number of items was reduced, the reliability and validity of the HIVSS-M-3 were examined by CTT and Rasch analysis.All self-reported information was collected online through the REDCap system, a web-based survey tool that is supported through the Clinical and Translational Science Institute (CTSI). Participants completed the 30-min REDCap survey, which included standardized measures to assess demographics, the HIV stigma scale, the Medical Outcomes Study–Social Support Survey , and the Center for Epidemiological Studies Depression Scale . The demographic variables included participant age, gender, marital status, ethnicity, educational level, employment status, health insurance, years of living with HIV, and recent CD4 and viral load. After completing the survey, participants were reimbursed for their participation.p < 0.05 was considered significant.Data analyses were conducted using IBM SPSS 23.0  and WINSTEPS 3.75.0 . Missing data were replaced using the multiple imputation calculation; p < 0.05) [We first conducted item analyses and deleted an item if it met the following criteria of CTT and Rasch analysis: (a) cross-loading or factor loading < 0.4 , (b) inf < 0.05) .After item reduction, we evaluated the following reliability and validity of the HIVSS-M-3 according to the recommendations in the COSMIN checklist .We used the COSMIN checklist with a 4-point scale to measure which of the descriptions on the translated scale adequately reflected the items from the original scale .We combined the exploratory factor analysis (EFA) in CTT and multidimensional Rasch analysis to assess the structural validity of the scale. In the EFA, principal component analysis (PCA) and oblique rotation were used. The number of factors were extracted based on the findings of parallel analysis . In multWe estimated the convergent validity of the HIVSS-M-3 by Pearson’s correlations, with expected significant positive correlation with the CES-D and negative correlation with the MOS-SSS.We used Cronbachs’ α and corrected item-total correlation to assess the internal consistency of the HIVSS-M-3 .Floor effects were evaluated by examining the percentage of the respondents that achieved the lowest possible scores. Ceiling effects were evaluated by examining the percentage of respondents that reached the highest possible score.Of the 216 PLWHA participants, 156 (72.22%) completed the questionnaires. The mean age of participants was 28.92 years (SD = 17.32) and the average years of living with HIV was 9.57 years (SD = 5.71). The average recent CD4 count was 683.49 (SD = 475.15), and the average viral load was 615.80 (SD = 1058.55). Table We found that the factor loading of item I-11 was less than 0.4, seven items  were cross-loading, and infit and outfit mean squares of three items  were outside the range of 0.6 to 1.4. In addition, two items  had a significant DIF across gender, and three items  had a significant DIF across education. According to the criteria of item retention, 12 items were deleted  met the requirements of “good” in the COSMIN checklist. We conducted the pilot test and formal survey to evaluate the items’ fluency, readability, and comprehensibility; all participants reported a good understanding of each item of the stigma scale.2 = 3672.360, df = 595, p < 0.001; Kaiser-Meyer-Olkin = 0.908). Based on parallel analysis, six factors were extracted with an eigenvalue of 2.00 to 6.18, together explaining 68.23% of the overall variance. Factor loadings for all items were between 0.46 and 0.86  and negative correlation with the MOS-SSS .The convergent validity for the HIVSS-M-3 was confirmed with positive correlation with the CES-D .The Cronbach’s alpha for the total HIVSS-M-3 was 0.95. The corrected item-total correlation ranged from 0.34 to 0.74  achieved the lowest possible score (35). No participant (0%) achieved the highest possible score on the scale (140). The lowest or highest possible scores were both below 15%, indicating that there were no floor or ceiling effects of the Myanmar version of the HIV stigma scale .The present study is one of the first examinations of the constructs of HIV stigma in the Myanmar context. The Myanmar version of the HIV stigma scale was adapted and validated through a rigorous, multiphase process that followed the guidelines prescribed in the Translation and Cultural Adaptation - Principles of Good Practice . Our psyThe factor analytic strategies used in CTT yielded a clear six-factor structure for the 35-item HIVSS-M-3. This finding confirmed that stigma differs as a construct across cultures , 9, 27.The HIVSS-M-3 was adapted from the Berger HIV stigma scale  and the On the other hand, considering the culturally specific characteristics of stigma, we also adapted 7 items of the HIV stigma scale tested in India. The EFA showed that 7 items were related to the PLHWAs’ religious concerns related to HIV and to possible vicarious stigma from healthcare providers . The religious-concerns finding reflects the importance of traditional religious theology regarding accepting one’s fate among PLWHA in Myanmar. The people in Myanmar are a culturally diverse population, with 74.4% of them being Buddhist, 8.2% a Christian, 3.8% a Muslim, 1.7% Hindu, 1.5% Confucianists, and 9.5% Ethnoreligionists . With thIn addition to the traditional CTT methods, the structural validity of the HIVSS-M-3 was also confirmed by Rasch analysis. Our data support that the category rating scale of the HIVSS-M-3 worked well and was free of DIF by gender and educational levels. The combination of a good person-separation index (> 2) and person reliability (> 0.8) suggested that the HIVSS-M-3 has acceptable measurement precision and is sensitive to distinguishing both high and low stigma participants .Similar to previous studies , 22, 27,This study has several limitations. First, the sample size was relatively small and some psychometric characteristics of the HIVSS-M-3 could be assessed further, such as test-retest reliability and structural validity, and these could be checked by confirmatory factor analysis. Second, the 37.6% non-response rate and the computer literacy of participants in responding to a self-assessment survey may impact the generalizability of the findings. Third, the REDCap online platform that was used also could potentially impact the generalizability of the findings. That is, persons who could not access such a survey or who are not literate in using online surveys were not included in this study. Finally, the sensitivity of the HIVSS-M-3 was not assessed. Therefore, future longitudinal or experimental studies are warranted for checking that. A further refinement and testing of the scale using a larger representative sample would produce more stable parameter estimations and robust results.The Myanmar version of the 35-item HIV stigma scale with a six-factor structure is a sufficiently valid and reliable tool for assessing the experience and effects of stigma in PLWHA in Myanmar. Furthermore, this stigma scale could also facilitate the development of stigma-reduction interventions and evaluate the effects of such interventions.Evidence has consistently indicated that HIV stigma is a common barrier to HIV prevention, testing, and treatment adherence . EspeciaAdditional file 1: Appendix A. Item and factor analysis of the Berger HIV stigma scale. Appendix B. Item and factor analysis of the 7-item stigma scale tested in India. Appendix C. The HIV stigma scale in Myanmar.


The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His Bile acids (BAs) are hydroxylated steroids synthesized by liver cells via a complex multistep process relying on the cytochrome P450-mediated oxidation of cholesterol ,2. BAs aThe trafficking of BAs is mediated by intracellular lipid-binding proteins (iLBPs), soluble carriers characterized by an internal cavity hosting hydrophobic or amphipathic molecules ,6. The iInherited and acquired defects of BA transporters are related to the pathogenesis of various hepatobiliary, intestinal and metabolic disorders . Thus, uE. coli cells as His6-tag protein. By the introduction of this affinity tag, we were able to raise the production yield and simplify the purification procedure. The recombinant cL-BABP showed an increased propensity to crystallize, allowing us to obtain its structure at high resolution. To validate the use of recombinant cL-BABP for structural investigations aimed at the development of innovative systems, our structures of cL-BABP and its complex with CA were compared with those formerly reported on the liver-extracted protein [For this purpose, we focused on the production of the recombinant cL-BABP and its characterization through X-ray crystallography. Unlike previous reports in the literature ,33, the  protein . This co protein ,30,31,32E. coli recombinant protein expression [E. coli [−1 bacterial culture. In order to simplify the purification protocol, minimizing the loss of protein due to multiple purification steps, we cloned the cL-BABP cDNA in an expression vector (pET15b) containing a single His6-tag at the protein N-terminus removable by means of thrombin cleavage , temperatures (25 °C and 32 °C), and incubation intervals (4 and 24 h). The best condition turned out to be 32 °C, 0.5 mM IPTG, and 4-h incubation, which produced the highest amount of protein in the soluble cellular fraction. The isolation of the target was then achieved through a purification procedure relying on two stages of nickel-affinity chromatography by means of a step-gradient elution protocol. After the first stage, a highly pure sample of His6-tag-cL-BABP was obtained. The second chromatographic step, performed after tag cleavage by thrombin protease, was done to obtain the pure mature cL-BABP. The final yield achieved through these simple procedures was improved to about 150 mg L−1 bacterial culture, allowing us to obtain a large amount of pure and homogeneous protein to perform X-ray crystallographic studies.Different protocols have been previously described to obtain purified cL-BABP both from its natural source, the chicken liver, and from pression ,27,33. Acleavage . To optiE. coli cells resulted in crystal growth using more than 30 different precipitant solutions. At variance with the liver-extracted protein, our recombinant cL-BABP displayed a higher propensity to crystallize. Indeed, crystal growth was observed both at room temperature and at 8 °C in a wide range of pH (spanning from 4.6 to 8.5) and with various combination of salts, organic acids, alcohols, and PEGs , as precipitant agents  . Crystalric unit . The ligric unit a. The twric unit b. CA-1 eThe CA-1 hydroxyls 7 and 12  vector was kindly provided by Dr. J. Foote . To generate cL-BABP in frame with the HisE. coli strain. Expression trials were performed to optimize the protein production by testing different IPTG concentrations (0.5 and 1 mM), temperatures (20 °C and 32 °C), and incubation intervals (4 and 24 h). The expression levels of the target protein were evaluated by SDS-PAGE analysis and compared to determine the conditions resulting in the maximal protein production in the soluble cellular fraction. In the best condition identified, bacterial cells were cultured in LB medium containing 100 mg L−1 ampicillin at 32 °C. cL-BABP over-expression was obtained after 4 h of induction at 32 °C in the presence of 0.5 IPTG when the OD600nm reached values between 0.6–0.8. Cells, harvested by centrifugation, were resuspended in buffer A  containing 20 mM imidazole and lysozyme (0.4 mg mL−1) and disrupted by sonication after 1 h of incubation on ice. The soluble cellular fraction, separated by centrifugation , was purified by nickel-affinity chromatography , using a step-gradient elution protocol relying on imidazole as a competitive agent. His6-tag cL-BABP elution was achieved at imidazole concentrations ranging from 75 to 250 mM. Fractions containing the target protein, identified by SDS-PAGE, were pooled and extensively dialyzed in buffer A. The His6-tag cleavage (−1 protein) directly inside the dialysis membrane. After 24 h, the tag cleavage was almost complete , and the mature protein was subjected to a second stage of nickel affinity chromatography. The effective tag removal was confirmed by Western blotting analysis using a HRP-conjugated His-tag monoclonal antibody . The high purity of the mature cL-BABP  was introduced by thermal shock in the BL21-Gold cleavage  was perf cL-BABP  obtained−1 in 100 mM NaCl and 20 mM TRIS, pH 7.5, and stored at 8 °C until required. Crystallization trials were performed on this protein sample using the vapor diffusion sitting drop technique either at 8 °C or at room temperature [v/v of either glycerol or ethylene glycol. Through this screening, we were able to identify the conditions yielding the crystals with the strongest diffraction patterns  solution, and equilibrated over a 500 μL reservoir. Crystals, grown within 1 day, were transferred to the cryoprotectant solution (20% v/v glycerol and 80% v/v precipitant) and flash-frozen in liquid nitrogen. The characterization of cL-BABP-CA complex was also attempted through the soaking technique by exposing pre-formed protein crystals to the ligand. 0.25 μL of a 10 mM solution of CA was added directly into the crystallization drop  and crystals were frozen in liquid nitrogen after a 2-h exposure. The recombinant mature cL-BABP  was concperature . More thMotion Determination web server [All crystals of cL-BABP and its complex with CA were screened for diffraction using synchrotron radiation at the Diamond Light Source  beamline I04. Full datasets were collected at 100 K on the Eiger2 XE 16M detector only on the crystal showing the strongest diffraction pattern . Reflectb server , resultib server  and PROCb server . Structub server . Data co6-tag cL-BABP we were able to improve the production yield to about 150 mg L−1 obtaining a large amount of pure and homogeneous protein through a simple purification procedure relying only on affinity chromatography. This protocol speeds up and simplifies the procedures for obtaining cL-BABP with respect to the extraction from liver cells and to former reports on recombinant protein expression in E. coli bacterial host [In this investigation, we report novel and effective protocols for the expression, purification, and crystallization of recombinant cL-BABP. By means of target expression as Hisial host ,33. The ial host  shows hiial host ,30,31,32